m*****n 发帖数: 760 | 1 本人对siRNA的设计和应用几乎无知,问题比较菜,请大家不要见笑。
需要用siRNA来knockdown一个外源表达的fusion protein(GFP-GOI),
因为不想影响endogenous GOI,所以想请教能不能设计siRNA只针对GFP,而不是GOI?
这样的siRNA会有效果吗?
另外大家都用什么program来设计siRNA,另外在哪家公司买siRNA啊?
谢谢了。 |
m*********7 发帖数: 5207 | 2 能不能设计siRNA只针对GFP,而不是GOI?
这样的siRNA会有效果吗? |
a***e 发帖数: 1010 | 3 ??
【在 m*********7 的大作中提到】![](/moin_static193/solenoid/img/up.png) : 能不能设计siRNA只针对GFP,而不是GOI? : 这样的siRNA会有效果吗?
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m*********7 发帖数: 5207 | 4 What's your question?
【在 a***e 的大作中提到】![](/moin_static193/solenoid/img/up.png) : ??
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a***e 发帖数: 1010 | 5 I guess you can silence GFP-GOI transgene by only targeting GFP region
without affecting endogenous GOI expression. |
m*****n 发帖数: 760 | 6 can you please explain more why targeting GFP only does not
interfere with the translation of the fusion protein?
Thank you.
【在 m*********7 的大作中提到】![](/moin_static193/solenoid/img/up.png) : 能不能设计siRNA只针对GFP,而不是GOI? : 这样的siRNA会有效果吗?
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m*****n 发帖数: 760 | 7 if this works, that would be exactly what I want.
in this way, I can determine if the phenotypes
are induced specificly by GFP-GOI transgene.
【在 a***e 的大作中提到】![](/moin_static193/solenoid/img/up.png) : I guess you can silence GFP-GOI transgene by only targeting GFP region : without affecting endogenous GOI expression.
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a****k 发帖数: 1130 | 8 这样是可以的
【在 m*****n 的大作中提到】![](/moin_static193/solenoid/img/up.png) : if this works, that would be exactly what I want. : in this way, I can determine if the phenotypes : are induced specificly by GFP-GOI transgene.
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H****H 发帖数: 11039 | 9 外源表达的干嘛需要knockdown啊?
你不转到细胞里不就行了么?
要用siRNA knockdown GFP-GOI而不影响GOI表达当然可以做到,设计target GFP序列的
siRNA就可以了。
【在 m*****n 的大作中提到】![](/moin_static193/solenoid/img/up.png) : 本人对siRNA的设计和应用几乎无知,问题比较菜,请大家不要见笑。 : 需要用siRNA来knockdown一个外源表达的fusion protein(GFP-GOI), : 因为不想影响endogenous GOI,所以想请教能不能设计siRNA只针对GFP,而不是GOI? : 这样的siRNA会有效果吗? : 另外大家都用什么program来设计siRNA,另外在哪家公司买siRNA啊? : 谢谢了。
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O******e 发帖数: 4845 | 10 他应该是想用siRNA去除GFP-GOI to verify their GFP signals (localization
pattherns, maybe?).
【在 H****H 的大作中提到】![](/moin_static193/solenoid/img/up.png) : 外源表达的干嘛需要knockdown啊? : 你不转到细胞里不就行了么? : 要用siRNA knockdown GFP-GOI而不影响GOI表达当然可以做到,设计target GFP序列的 : siRNA就可以了。
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m*****n 发帖数: 760 | 11 we want to determine if the phenotypes of the transgenic animals
are specifically caused by GFP-GOI transgene.
if by knocking down GFP-GOI transgene only, but not endougeneous GOI,
the phenotypes are gone or less severe, then we are more confident that
ectopic expression of GOI induces the phenotypes.
【在 O******e 的大作中提到】![](/moin_static193/solenoid/img/up.png) : 他应该是想用siRNA去除GFP-GOI to verify their GFP signals (localization : pattherns, maybe?).
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m*********7 发帖数: 5207 | 12 In theory it should work, but then you have to make sure it is not due to
off-target effect, and later rescue the phenotype by making RNAi resistant
GFP-GOI to demonstrate specificity. What's more, what if the effect of
endogenous and ectopic GOI are not additive or synergistic?
【在 m*****n 的大作中提到】![](/moin_static193/solenoid/img/up.png) : can you please explain more why targeting GFP only does not : interfere with the translation of the fusion protein? : Thank you.
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